[논문]Structural Insight into Dihydrodipicolinate Reductase from Corybebacterium glutamicum for Lysine Biosynthesis

Sagong, Hye-Young; Kim, Kyung-Jin

doi:10.4014/jmb.1508.08086

Structural Insight into Dihydrodipicolinate Reductase from Corybebacterium glutamicum for Lysine Biosynthesis 원문보기

Journal of microbiology and biotechnology, v.26 no.2, 2016년, pp.226 - 232

Sagong, Hye-Young (School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University) , Kim, Kyung-Jin (School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University)

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Dihydrodipicolinate reductase is an enzyme that converts dihydrodipicolinate to tetrahydrodipicolinate using an NAD(P)H cofactor in L-lysine biosynthesis. To increase the understanding of the molecular mechanisms of lysine biosynthesis, we determined the crystal structure of dihydrodipicolinate reductase from Corynebacterium glutamicum (CgDapB). CgDapB functions as a tetramer, and each protomer is composed of two domains, an Nterminal domain and a C-terminal domain. The N-terminal domain mainly contributes to nucleotide binding, whereas the C-terminal domain is involved in substrate binding. We elucidated the mode of cofactor binding to CgDapB by determining the crystal structure of the enzyme in complex with NADP+ and found that CgDapB utilizes both NADH and NADPH as cofactors. Moreover, we determined the substrate binding mode of the enzyme based on the coordination mode of two sulfate ions in our structure. Compared with Mycobacterium tuberculosis DapB in complex with its cofactor and inhibitor, we propose that the domain movement for active site constitution occurs when both cofactor and substrate bind to the enzyme.

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Crystallization of the purified CgDapB protein was initially performed with commercially available sparse-matrix screens from RIGAKU and Molecular Dimensions using the hanging-drop vapor-diffusion method at 20℃. Each experiment consisted of mixing 1.
The structure of apo-form of CgDapB was determined by molecular replacement with the CCP4 version of MOLREP [19], using the structure of DapB from Mycobacterium tuberculosis (PDB code 1YL5) as a search model. Further model building was performed manually using the program WinCoot [3], and refinement was performed with CCP4 refmac5 [12] and CNS [1].

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4B). Based on these observations, we propose that CgDapB shares both the catalytic mechanism and substrate binding mode with MtDHPR.
It has been reported that MtDHPR undergoes an open/closed domain movement upon binding of the cofactor and substrate. Interestingly, we did not observe domain movement upon binding of the NADP+ cofactor, but rather the position of NTD in the apo-form was nearly identical to that in the NADP+-bound form (Fig. 5).
The CgDapB tetramer was generated by crystallographic symmetry operation, and the results of size-exclusion chromatography confirmed that CgDapB functions as a tetramer (data not shown). The tetramer is exclusively constituted by interactions between the CTDs.
The crystal belonged to the I4122 space group, and the asymmetric unit of the crystal contained two CgDapB molecules. The overall structure of CgDapB was homologous to that of dihydrodipicolinate reductase from Mycobacterium tuberculosis (MtDHPR), and the amino acid sequence identity between CgDapB and MtDHPR was 61% (Fig. 2A).
To identify the NADP+ binding mode, we determined the crystal structure of CgDapB in complex with the NADP+ cofactor at 2.12 Å resolution. When we superimposed the NADP+-bound form of CgDapB with the apo-form of the enzyme, regional structural changes were observed, although overall the two structures were nearly identical.
We could not determine the CgDapB structure in complex with the dihydrodipicolinate substrate; however, we can infer the substrate binding mode of the enzyme from the MtDHPR structure in complex with 2,6-pyridinedicarboxylate (2,6-PDC), a competitive inhibitor of DHPR. In MtDHPR, the substrate binding site is located in the CTD; the aromatic ring of 2,6-PDC is stacked against the nicotinamide ring of bound NADP+ cofactor, and two carboxyl groups form hydrogen bonds with neighboring residues such as His133, Lys136, and Thr143.
To determine the molecular mechanism of dihydrodipicolinate reductase from C. glutamicum (CgDapB), we purified, crystallized, and determined the crystal structure of the enzyme at 2.5 Å resolution (Fig. 1B).

참고문헌 (20)

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Structural Insight into Dihydrodipicolinate Reductase from Corybebacterium glutamicum for Lysine Biosynthesis 원문보기

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Structural Insight into Dihydrodipicolinate Reductase from Corybebacterium glutamicum for Lysine Biosynthesis 원문보기

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